Cellular nuclei of new blood vessels protruding into the vitreous cavity were also observed in TeM-injected retinas by immunohistochemistry. Western blotting revealed a 16-kDa immunoreactive protein, indicating incorporation of an exogenous TeM fragment into the retina. Our data shows that TeM can effectively inhibit pathological angiogenesis in mouse eyes; indicating its potential role in prevention and treatment of ocular neovascularization.We aimed to determine the anti-angiogenic effect of tenomodulin (TeM) on retinal neovascularization in an oxygen-induced retinopathy (OIR) mouse model. OIR was induced in C57BL/6 mice by exposing seven-day-old mice to 75% oxygen for five days followed by room air for five days. Control mice were exposed to room air from birth until postnatal day 17. Mice received intravitreal injections of 1 μg of TeM in one eye and PBS in the contralateral eye at P7 before being exposed to 75% oxygen. Eyes were collected at postnatal day 17. Retinal blood vessel patterns were visualized by fluorescein angiography. We quantified the number of neovascular nuclei that were present beyond the inner limiting membrane (ILM) using histological methods with a masked approach. Furthermore, double immunohistochemical staining of TeM was performed on retinas to identify nuclei protruding into the vitreous cavity. Western blot was used to detect exogenous TeM protein. The central nonperfusion area (NPA, mm Retinal neovascularization, the pathological growth of new blood vessels, is characteristic of several eye diseases that cause catastrophic vision loss throughout the world. Thus, the control of ocular neovascularization is promising for the treatment of these disorders ,2. Howevin vitro, and that it suppresses tumorigenesis in vivo[in vivo has not been extensively studied. In this study, we explored the anti-angiogenic potential of TeM on retinal neovascularization in a mouse model, using intravitreal administration.Tenomodulin , a novel member of the tumor necrosis factor family , has bees in vivo,26–28. Hp < 0.001), and the PBS-injected contralateral control eye (p < 0.001). There were also very few abnormal vessels, although signs of central vaso-obliteration and neovascularization were still present  in the means among TeM-injected mice and OIR mice, and TeM-injected eyes and PBS-injected control eyes. Thus, prophylactic injection of TeM significantly attenuated the neovascularization response.For quantitative assessment of the vasoproliferation response, HE staining was performed, and the nuclei of new blood vessels extending beyond the inner limiting membrane (ILM) into the vitreous body were counted. An average of 15–18 sections for each group was assessed. Either no neovascular nuclei or very few nuclei were found in retinas of mice raised in the normoxic condition , whereasThe nuclei of new blood vessels protruding from the inner retina into the vitreous cavity were observed in double immunohistochemical-stained frozen retinal sections of P17 C57BL/6 mice . CD31 seExogenous TeM protein was detected by western blot analysis as a prominent band of approximately 16 kDa (which is its expected size) in extracts from P17 TeM-injected retinas. This demonstrates the presence of TeM used in the experiments. In contrast, no immunoreactive band was detected in extracts from PBS-injected control retinas . This deTeM (2 mg/mL) was generously provided by Dr. K. Yamana, Teijin Pharmaceutical Co., Ltd. . It was dissolved in a solution containing 25 mM HEPES and 0.15 M NaCl at pH 8.3, and stored at −80 °C. The TeM protein has a strong hydrophobic nature, high concentrations that result in precipitation, and freeze-thaw cycles can be repeated 2 to 3 times. The activity of the TeM protein can be maintained for 2 to 3 days in aqueous solution.This study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Neonatal mice (C57BL/6) were provided by Charles River Laboratories, Osaka, Japan and housed in the animal facility of the Osaka University Graduate School of Medicine. C57BL/6 mice were randomly divided into three groups: a normoxia group (control group), an oxygen-exposed group (OIR group), and a TeM group; each group had one nursing mother and five pups. The mice in the normoxia group were maintained in room air from birth until postnatal day 17 (P17). The mice in the oxygen-exposed group were exposed to 75% ± 2% oxygen (hyperoxia) at postnatal day 7 (P7) for five days and then returned to room air for another five days, producing relatively hypoxic conditions. In the TeM experimental group, 1 μg (0.5 μL) TeM was injected into the vitreous body at P7 with a 32 gauge needle  in one eye, while the same volume of PBS was injected into the other eye as a control. As soon as the injections were finished, the mice were then exposed to a 75% ± 2% oxygen environment.6 molecular weight fluorescein-dextran . Eyes from the normoxia, OIR, and intravitreal injection groups were enucleated and placed in 10% formalin neutral buffer for 4 to 24 h. Corneas and lenses were removed; peripheral retinas were cut and then flat-mounted on a glass slide with glycerol-gelatin. The flat-mounted retinas were viewed by fluorescence microscopy  and photographed. The central nonperfused area of the retina was quantified from the digital images in a masked fashion, using image processing and analysis software .Fluorescein angiography of the retina was performed as previously described . On P17,Mice from all groups were sacrificed on P17. Eyes were removed and fixed with 4% paraformaldehyde in PBS for 4 h followed by immersion in 20% sucrose in PBS overnight for frozen sections. The tissues were embedded in optimal cutting temperature (OCT) compound , frozen at −80 °C, and sectioned at an 8-μm thickness with a cryostat at −20 °C. The sections were taken at roughly 100 μm intervals, spanning the entire retina. Eight-micrometer thick frozen tissue sections adjacent to those used to quantitate retinal neovascularization were stained with Mayer’s hematoxylin (5 min) and eosin (3 min). Specimens were then dehydrated and cover-slipped. Photographs were taken to quantify the neovascular response. Eyes of 5–8 mice from each group were analyzed . For each eye, three sections were randomly chosen and evaluated, neovascular cell nuclei protruding into the vitreous from the ILM were counted using a masked protocol, and the mean number of neovascular nuclei per section was determined.Mice from all groups were sacrificed on P17. Eyes were removed and fixed with 4% paraformaldehyde in PBS for 4 h followed by immersion in 20% sucrose in PBS overnight for frozen sections. The tissues were embedded in OCT compound , frozen at −80 °C, and sectioned at an 8-μm thickness with a cryostat at −20 °C. Sections were obtained at roughly 100 μm intervals, spanning the entire retina. After three 5-min washes with PBS, sections were incubated for 1 h with 5% normal donkey serum at room temperature, and were then incubated overnight at 4 °C with affinity purified goat polyclonal anti-mouse IgG  as the primary antibody, diluted 1:50 with 1% normal donkey serum in PBST (0.3% Triton X in PBS). For the control, normal goat IgG  was used as the primary antibody. Sections were rinsed in PBS 3 times (5 min each time) and incubated with a secondary Alexa 488 donkey anti-goat IgG-conjugated antibody  plus DAPI  for double-staining for 1 h at room temperature. After rinsing the sections again, they were mounted with glycerol-gelatin, viewed under a fluorescence microscope , and photographed.Intravitreal-injected retinas from P17 mice were dissected and lysed in RIPA buffer  with proteinase inhibitor on ice for gel analysis. Samples were homogenized and then centrifuged at 1.5 K rpm for 5 min at 4 °C. Supernatant was loaded and electrophoresed on 12.5% SDS-poly-polyacrylamide gel and transferred to Hybond-P membranes . After preincubation with blocking buffer (5% BSA in PBST) at 4 °C overnight, the membrane was incubated with anti-FLAG M2 monoclonal antibody  for 30 min at room temperature. After washing, HRP-conjugated rabbit anti-mouse IgG  was added for 30 min at room temperature with shaking, according to the manufacturer’s protocol. Visualization was achieved using DAB substrate buffer and DAB chromogen .p values of <0.05 were considered statistically significant.Each experiment was performed at least two times, and the data were analyzed statistically using Statistical Package for the Social Sciences (SPSS 13.0), using one-way analysis of variance (ANOVA), followed by Scheffé’s pairwise comparison. N-terminal region, 2 N-linked glycosylation sites, and a cysteine-rich domain  in the C-terminal region [C-terminal region, which corresponds to the predicted anti-angiogenic functional domain of ChM-I  [C-terminal domains, and the spacing of the 8 cysteine residues in TeM is almost identical with that in ChM-I [C-terminal characteristics between TeM and ChM-I, and the structural analogy noted between skeletal connective tissues and the eye in terms of hypovascularity and the components of their extracellular matrix, it is possible that TeM also participates in the maintenance of avascular conditions in the eye.Tenomodulin is a member of a new family of type II transmembrane glycoproteins ,26. It il region ,23,24. Tin ChM-I ,23. Howein ChM-I , the TeMin ChM-I ,23. Becain vitro and have a strong anti-tumor effect in vivo. Clinical and laboratory studies have also reported robust evidence indicating that tumor angiogenesis is inhibited by administration of anti-angiogenic factors. Oshima et al. have also reported that recombinant ChM-I protein effectively suppresses the growth of chondrosarcoma and colon adenocarcinoma by inhibiting angiogenesis in vivo[C-terminal ChM-I-like domain of TeM has anti-tumor activity in vivo, and indeed expression of both Ad-shTeM  and Ad-shChM-I  result in the effective inhibition of angiogenesis, followed by suppression of tumor growth [Both TeM and ChM-I have been reported to inhibit proliferation and tube morphogenesis of vascular endothelial cells s in vivo. It is lr growth . The conin vivo also remains unclear. Previously, Docheva et al. reported that mice lacking both TeM and ChM-I showed normal OIR retinal vasculature, and that double-deficient mice did not have changes in tendon vessel density. This suggested that TeM is a regulator of tenocyte proliferation and is involved in collagen fibril maturation, but did not support a role for TeM in angiogenesis in vivo[in vitro and in vivo studies regarding the role of ChM-I and TeM in angiogenesis led us to investigate TeM function in retinal neovascularization, which had not been studied in oxygen-induced retinopathy (OIR) in C57BL/6 mice. In the present study, we used a highly reproducible mouse model of ischemia-induced retinopathy [C-terminal fragment of TeM after cleavage at a predicted site [C-terminal of full-length TeM is exposed on the cell surface [C-terminal domain of both TeM and ChM-I exhibits antiangiogenic properties [The anti-angiogenic potential of TeM on retinal neovascularization s in vivo. The coninopathy . This moinopathy . As demoinopathy ,25,26. Win vivo remains unclear. Previous studies, both in vitro and in vivo, have demonstrated that VEGF plays a major role in stimulating retinal neovascularization [C terminus of TeM restricting vascular formation in the retina. The fact that TeM is a transmembrane protein and the presence of its antiangiogenic domain on the cell surface may work as an antiangiogenic barrier for the physiological prevention of vascular invasion into hypovascular tissues [C-terminal fragments of TeM and ChM-I, both of which are located downstream of the transmembrane domain, are responsible for their antiangiogenic activity [The mechanism of inhibition of pathological angiogenesis by TeM rization ,8,30–34.rization . Many strization ,35–37. Trization ,35,38. Irization ,39. Thesrization ,20,28. I tissues . On the The findings of this study indicate that TeM is effective, at least in part, in preventing pathologic angiogenesis in a mouse model of ischemic retinopathy. A better understanding of the biology and mechanism of action of TeM may someday lead to the development of novel and more effective therapeutic approaches for the treatment of pathologic neovascular conditions. Thus, these findings offer preventive and therapeutic potential for the management of pathologic intraocular angiogenesis.


The supersaturatable self-microemulsifying drug delivery system (S-SMEDDS) of CBZ was evaluated in-vitro and in-vivo. Three different formulations of CBZ were prepared and drug precipitation behavior, dissolution rate in-vitro and particle size distribution were evaluated. Studies on CaCO-2 permeability of three formulations were also carried out. Pharmacokinetic studies were conducted in beagle dogs with administration dose of 200mg to assess bioavailability in-vivo compared with commercial tablet. The results showed that the presence of a small amount of polymeric precipitation inhibitor (PVP) effectively sustained supersaturated state by retarding precipitation kinetics. The mean particle size after dispersion was about 33.7 nm and the release rate from S-SMEDDS was significantly higher than the commercial tablet in-vitro. S-SMEDDS formulation with precipitation inhibitor decreased impairment to cells due to a lower surfactant level compared to SMEDDS. The absorption of S-SMEDDS in-vivo resulted in about 5-fold increase in bioavailability compared with the commercial tablet and the reproducibility of plasma concentration profiles intra-individual was improved remarkably. This study demonstrates that S-SMEDDS technology provide an effective approach for improving the extent of absorption of poorly-soluble drugs with low level of surfactant.The supersaturatable self-microemulsifying drug delivery system (S-SMEDDS) represents a new thermodynamically stable formulation approach wherein it is designed to contain a reduced amount of surfactant and a water-soluble polymer (precipitation inhibitor or supersaturated promoter) to prevent precipitation of the drug by generating and maintaining a supersaturated state  CBZ tβ-CD) , 10.in-vivo. The S-SMEDDS formulations can result in enhanced oral absorption as compared with the related self-emulsifying drug delivery systems (SMEDDS) formulation and the reduced surfactant levels may minimize gastrointestinal surfactant side effects (Self-microemulsifying drug delivery system (SMEDDS) can be defined as an isotropic multi-component drug delivery system composed of surfactant, co-surfactant and oil, which spontaneously form microemulsion in the presence of water -12. Conv effects -20.in-vitro and bioavailability as well as reducing the intra- and inter subject variability of the blood level of CBZ in-vivo.In S-SMEDDS, solubilization of CBZ in o/w microemulsions will enhance its bioavailability with low surfactant level and CBZ can be absorbed in GI homogeneously, which may reduce variability in plasma concentration. The main objectives of the study were to develop S-SMEDDS of CBZ to improve its dissolution Materials and apparatusAPI of CBZ was purchased from Jiuzhou pharmaceutical Industrial Co.(China), Tween 80 and PEG400 were obtained from sigma Chem. Co. and medium chain triglycerides (MIGLYOL 812N) were supplied by Gatte-fosse (France); Cremphor EL-35 was from BASF (Germany), and PVPK30 was from ISP (USA). S-SMEDDS of CBZ were prepared by our lab and CBZ tablets were purchased from Shanghai Sanlian Pharma Ltd. (batch No.20061101). Beagle dogs were from animal center of Academy of Military Medical Sciences 13-15 Kg male.Hermle Z200A high speed centrifugator was from Germany, Hitachi L-7100, HPLC was from Japan, QL-901 vortex mixer came from Shanghai, China UV-160A vis-ultraviolet spectrophotometer was from SHIMAZU Co. Tokyo, Japan, and ELECTROLAB TDT-08L drug dissolution test instrument was made in ELECTROLAB corporation, U.S.A.Different formulations of CBZThree formulations with or without precipitation inhibitor were prepared as showed in Formulation A (SMEDDS) with high level of surfactant without precipitation inhibitor, Formulation B (S-SMEDDS) with low level of surfactant with 2% (w/w) precipitation inhibitor and Formulation C (SMEDDS) with low level of surfactant without precipitation inhibitor were compared and evaluated.In-vitro evaluation of precipitationin-vitro. One gram of the S-SMEDDS formulations was placed into 50 mL of the medium and stirred at 50 r/min. About 0.5 mL solution samples were taken at 0, 4, 8, 12, 24 and 36 h and the filtrate were analyzed by HPLC at wavelength of 285 nm.The performance of three formulations of CBZ was evaluated with respect to drug precipitation when contacted with an aqueous medium. 0.1 mol/L HCl was chosen as the medium Dissolution in-vitroA CP2005 apparatus with baskets rotating at 50 r/min was used. A volume of 0.1 mol/LHCl (900 mL) was used as the dissolution medium and maintained at 37 ± 0.5°C.The market tablets and S-SMEDDS formulation of CBZ ( formulation B) in soft capsule were positioned in the basket at t = 0 min. Samples of 5 mL were withdrawn at various intervals of time  and the samples was filtered by 0.22 μm filter film. The volume of the dissolution medium was kept constant throughout the run by replacing the removed samples with an equal quantity of freshly solution. In all cases, three runs were carried out for each formulation. The accumulated amount of drug released at each sampling point was corrected with the volume of the dissolution medium. The concentration of CBZ in each samples were analyzed spectrophotometrically at wavelength of 285 nm.Emulsion droplet size analysisFormulation B-2 (1 g) was diluted with purified water at 37°C with a stirring rate of 50 r/m using a rotating paddle dissolution apparatus. The droplet size distribution of the resultant emulsions after 20 min was determined by a laser diffraction sizer , with the ability of measuring sizes between 10 and 5000 nm.CaCO-2 cell permeability studyCells culture and recovery2 were considered as recovered. The average TEER-values were 654 ± 43Ω•cm2.CaCO-2 cells, originating from a human colorectal carcinoma, were provided by ATCC (American Type Culture Collection). For these transport studies, cells at passage 50 were used and allowed to grow and differentiate to confluent monolayer for 21 days. Confluence and differentiation of the cell monolayer on inserts was measured through evaluation of the Trans-Epithelial-Electrical-Resistance (TEER). . Inserts containing PBS buffer ( pH = 7.2) alone served as a negative control. At the end of the experiment, PBS was replaced by growth medium. Inserts were incubated for an additional 48 h for recovery TEER measurements. Inserts with TEER-values > 600Ω•cmPermeability studiesPrior to the experiment, the culture medium was removed, cells were washed with PBS and incubated for 30 min at 37°C, 45 r/m. The filter-grown monolayers of 21–23 days of age were exposed to different CBZ S-SMEDDS  within aPharmacokinetic behavior in beagle dogsThe study was approved by the Ethical Committee of academy of military medical sciences. Six beagle dogs, weighting 13-15 Kg, male, were obtained from animal center of academy of military medical sciences . All animals were housed individually in standard cages on a 12 h light-dark cycles and were fed with standard animal chow daily and had free access to drinking waterExperimental protocolAfter an overnight (12 h) fasting, six beagle dogs were administered orally with the dose of 200 mg per dog. According to Determination of CBZ concentration in plasmaThe mobile phase consisted of methanol-water , at a flow rate of 1 mL/min. The column used was ZORBAX SB-C18 .The detection wavelength was 285 nm. 50 μL of diazepam(10.0 μg/mL) internal standard solution was added to 0.5 mL plasma in a 10 mL glass centrifuge tube for assay and then 3 mL ethylether was added, the samples were shaken for 5 min by vortex mixer and centrifuged at 3000 r/min for 10 min. The supernatant phase was separated and evaporated to dryness at 40°C under nitrogen. Residues were dissolved in 100 μL mobile phase and were assayed by HPLC.Data analysis0~12h) was calculated by linear trapezoidal rule from zero to the last plasma concentration. The maximum plasma concentration, Cmax, and the time of its occurrence, Tmax, were compiled from the concentration–time data. All results were expressed as mean ± SD. The data from different formulations were compared for statistical significance by one way analysis of variance (ANOVA). T-tests were performed to evaluate the significant differences between the two formulations and the data were considered statistically significant at p < 0.05.Pharmacokinetic analysis was performed by means of a model independent method using DAS2.0 computer program (issued by the State Food and Drug Administration of China for pharmacokinetic study). The area under the plasma concentration versus time curve from zero to 12 h . This indicated that although the formulations affected the tightness of the cell monolayer, it reversibly recovered after the experiment. On the other hand, the TEER-value of monolayers reduced remarkably when treated with formulation A , and there was no significant differences between formulation B and formulation C (p > 0.05). From the results of permeability of CBZ across CaCO-2 cell mono-layers for 15, 30, 45 and 60 min and the corresponding peak time (tmax) were determined through the inspection of the individual drug plasma concentration-time profiles. AUC0-t was calculated by the linear trapezoidal rule, and AUC0-∞was calculated as AUC0-t+Ct / Ke. The results are showed in 0-t, and Cmax had significant differences between S-SMEDDS formulation and the commercial tablet. Plasma concentration-time curve of CBZ commercial tablet (reference) and S-SMEDDS (test) in beagle dogs (n = 6) can be seen in in-vivo resulted in about 5-fold increase in bioavailability compared with the conventional tablet and the reproducibility (RSD%) of plasma concentration profiles intra-individual was improved remarkably. The higher absorption characteristics of CBZ from the S-SEDDS formulation are attributed to a high free drug concentration in-vivo, implying a supersaturated state. So it was concluded that S-SMEDDS of CBZ enhanced its bioavailability with low surfactant level and CBZ can be absorbed in GI homogeneously which may reduce variability in plasma concentration. The maximum plasma concentration , supersaturated solubilized molecules or “compound” with precipitation inhibitor (interacted with excipients), molecules partitioned into microemulsions and precipitated solid particles. It is difficult to separate the drug in each phase; in this case, dissolved CBZ which was determined in the dissolution and precipitation test contains undissolved small droplets and particles through the 0.22 μm filter. On the other hand, the particle size of the precipitated solids grew significantly during the course; the actual degree of supersaturation at any given time was difficult to determine precisely.An In-vivo bioavailability in-vivo study for the comparison with conventional SMEDDS. From the evaluation results of precipitation in-vitro, SMEDDS formulation without precipitation inhibitor at low surfactant level was not stable with occurrence of drug precipitation within 4 h. S-SMEDDS with the presence of a small amount of polymeric precipitation inhibitor (PVP) effectively sustained supersaturated state by retarding precipitation kinetics. When SMEDDS formulations are introduced into gastrointestinal area(GI), drug precipitation may occur and lead to failure of improvement of intestinal absorption. On the other hand, high surfactant level typically present in SMEDDS formulations can cause GI side-effects. The supersaturatable self-microemulsifying drug delivery system (S-SMEDDS) represents a new thermodynamically stable formulation approach wherein it is designed to contain a reduced amount of surfactant and a water-soluble polymer (precipitation inhibitor or supersaturated promoter) to prevent precipitation of the drug by generating and maintaining a supersaturated state in-vivo. In S-SMEDDS, solubilization of CBZ in o/w microemulsions will enhance its bioavailability with low surfactant level and CBZ can be absorbed in GI homogeneously which may reduce variability in plasma concentration.SMEDDS and S-SMEDDS are systems which can form fine oil−in−water (o/w) microemulsions in the gastrointestinal tract. This formation of fine droplets provides a large surface area for pancreatic lipase to hydrolyse triglycerides and thereby promots a rapid release of the drug substance and/or the formation of mixed micelles containing the drug substance. Greater availability of dissolved CBZ from the SMEDDS or S-SMEDDS formulation could lead to higher absorption and higher oral bioavailability. However, we have not performed in-vitro and bioavailability in beagle dogs in-vivo were evaluated. The results demonstrate that S-SMEDDS technology provide an effective approach for improving the extent of absorption of poorly-soluble drugs with low surfactant level and decreased impairment to cells compared to SMEDDS.S-SMEDDS of CBZ were developed and their drug precipitation behavior, CaCO-2 cell permeability, dissolution rate


Collectively these data propose a role for GLP-1 receptors in regulating drug reward. Moreover, the GLP-1 signaling system may be involved in the development of drug dependence since the rewarding effects of addictive drugs involves interferences with the mesolimbic dopamine system. Given that GLP-1 analogues, such as exenatide and liraglutide, are clinically available for treatment of type II diabetes, we propose that these should be elucidated as treatments of drug dependence.Glucagon-like peptide 1 (GLP-1) is an incretine hormone that controls consummatory behavior and glucose homeostasis. It is released in response to nutrient ingestion from the intestine and production in the brain has also been identified. Given that GLP-1 receptors are expressed in reward areas, such as the nucleus accumbens and ventral tegmental area, and that common mechanisms regulate food and drug-induced reward we hypothesize that GLP-1 receptors are involved in reward regulation. Herein the effect of the GLP-1 receptor agonist Exendin-4 (Ex4), on amphetamine- and cocaine-induced activation of the mesolimbic dopamine system was investigated in mice. In a series of experiments we show that treatment with Ex4, at a dose with no effect  Glucagon-like peptide 1 (GLP-1), an incretine gut-brain hormone, is released in response to nutrient ingestion . In addiUsage of psychostimulants, such as amphetamine and cocaine, may cause addiction and the addiction related costs are significant for both society and the individuals ,16. Drugad libitum, except during the experimental setups. The study was carried out in strict accordance with the recommendations in the Swedish Animal Welfare Act and all experiments were approved by the Swedish Ethical Committee on Animal Research in Gothenburg . All efforts were made to minimize animal suffering, and to reduce the number of animals used. All animals were allowed to acclimatize at least one week before the start of the experiments.Adult post-pubertal age-matched male NMRI mice  were used as described previously . In brieper se as well as blocked the rewarding properties of alcohol in mice [Dex-amphetamine sulphate  was dissolved in vehicle (0.9% sodium chloride solution) and was administered i.p. at a dose of 2 mg/kg 10 minutes prior to initiation of the experiment. Cocaine  was dissolved in the vehicle (0.9% sodium chloride solution) and was administered i.p. at a dose of 10 mg/kg 10 minutes prior to initiation of the experiment. Similar doses have been used previously to induce an activation of the mesolimbic dopamine system as measured by locomotor activity and accumbal dopamine release in rats . The pep in mice . In all Locomotor activity was recorded as described previously . In brieThe mice were allowed to habituate to the locomotor activity box one hour prior to drug challenge. In the first series of experiment the effects of Ex4  on amphetamine-induced  locomotor stimulation were investigated. Ex4 was administered ten minutes prior to amphetamine and the activity registration started ten minutes after the last injection. Each mouse received one treatment combination  and was only subjected to one experimental trial. In a subsequent series of experiment, in separate mice, the effects of Ex4  on cocaine-induced  locomotor stimulation were investigated. Ex4 was administered ten minutes prior to cocaine and the activity registration started ten minutes after the last injection. Each mouse received one treatment combination  and was only subjected to one experimental trial.For measurements of extracellular dopamine levels, mice were implanted unilaterally with a microdialysis probe positioned in the nucleus accumbens. The surgery was performed as described in detail previously . In brieIn the first series of experiments the effect of systemic administration of Ex4  on amphetamine-induced  accumbal dopamine release was investigated using microdialysis in freely moving mice. In subsequent experiments, in separate mice, the effects of Ex4  on cocaine-induced  accumbal dopamine release was investigated. As described below a similar procedure for both these series of experiments was used.On the test day the probe was connected to a microperfusion pump  and perfused with Ringer solution at a rate of 1.5 µl/minute. After one hour of habituation to the microdialysis set-up, perfusion samples were collected every 20 minutes. The baseline dopamine level was defined as the average of three consecutive samples before the first drug/vehicle challenge, and the increase in accumbal dopamine was calculated as the percent increase from baseline. After the baseline samples (-40 minutes until 0 minutes), mice were injected with Ex4 or vehicle (at 10 minutes), which was followed by an amphetamine or vehicle injection (at 20 minutes). In the second series of experiments mice were initially injected with Ex4 or vehicle (at 10 minutes), which was followed by a second injection containing either cocaine or vehicle (at 20 minutes). Following these drug administrations an additional eight 20 minute samples were collected. Collectively the following treatment groups (n=8 in each group) were created. In the first series of experiments: vehicle-vehicle (Veh-Veh), vehicle-amphetamine (Veh-Amph), Ex4-vehicle (Ex4-Veh) and Ex4-amphetamine (Ex4-Amph). In the second series of experiments: vehicle-vehicle (Veh-Veh), vehicle-cocaine (Veh-Coc), Ex4-vehicle (Ex4-Veh) and Ex4-cocaine (Ex4-Coc).The dopamine levels in the dialysates were determined by HPLC with electrochemical detection. A pump , an ion exchange column  and a detector  equipped with a VT-03 flow cell (Antec Leyden) were used. The mobile phase (pH 5.6), consisting of sulfonic acid 10 mM, citric acid 200 mM, sodium citrate 200 mM, 10% EDTA, 30% MeOH, was vacuum filtered using a 0.2 µm membrane filter . The mobile phase was delivered at a flow rate of 0.2 ml/minute passing a degasser , and the analyte was oxidized at +0.4 V.After the microdialysis experiments were completed, the mice were decapitated, and probes were perfused with pontamine sky blue 6BX to facilitate probe localization. The brains were mounted on a vibroslice device  and cut in 50 µm sections. The location of the probe was determined by gross observation using light microscopy. The exact position of the probe was verified  and onlyper se [To evaluate the effects of Ex4 on the rewarding effects of either amphetamine or cocaine, CPP tests were performed in mice as previously described . In brieper se .Condition place preference was calculated as the difference in % of total time spent in the drug-paired (i.e. least preferred) compartment during the post-conditioning and the pre-conditioning session.Locomotor activity data was evaluated by a one-way ANOVA followed by Bonferroni post-hoc tests. The microdialysis experiments were evaluated by a two-way ANOVA followed by Bonferroni post-hoc test for comparisons between different treatments and specifically at given time points. The condition place preference data were evaluated by an unpaired t-test. Data are presented as mean ± SEM. A probability value of P<0.05 was considered as statistically significant.P=0.0027; n=8 per group). As shown in P<0.01). This amphetamine-induced locomotor activity was significantly reduced by pre-treatment with a single injection of Ex4 (P<0.01), at a dose that alone had no significant effect on locomotor activity compared to vehicle treatment (P>0.05). There was no difference in locomotor activity response in vehicle treated mice and Ex4-Amph treated mice (P>0.05).An overall main effect of treatment was found on locomotor activity in mice following systemic administration of amphetamine (2 mg/kg) and Ex4 (2.4 µg/kg) =6.03, P>0.05). There was a significant difference in accumbal dopamine response in vehicle treated mice and Ex4-Amph treated mice at time interval 40-100 minutes (P>0.001).Accumbal microdialysis measurements of dopamine in mice revealed an overall main effect of treatment =206.50, P<0.0001), F=12.00, P<0.0001) and treatment x time interaction =10.65, P<0.0001)  (Amph-Veh) CPP was significantly attenuated by an acute single injection of Ex4 (2.4 µg/kg) (Amph-Ex4) on the post-conditioning day compared to vehicle injection . As shown in P<0.001). This cocaine-induced locomotor activity was significantly reduced by pre-treatment with a single injection of Ex4 (P<0.05), at a dose that alone had no significant effect on locomotor activity compared to vehicle treatment (P>0.05). There was no difference in locomotor activity response in vehicle treated mice and Ex4-Coc treated mice (P>0.05).An overall main effect of treatment was found on locomotor activity in mice following systemic administration of cocaine (10 mg/kg) and Ex4 (2.4 µg/kg) =9.63, P>0.05). There was a significant difference in accumbal dopamine response in vehicle treated mice and Ex4-Coc treated mice at time interval 40 minutes (P<0.05), 60 minutes (P<0.001) and 80 minutes (P<0.05).Accumbal microdialysis measurements of dopamine in mice revealed an overall main effect of treatment =95.00, P<0.0001), time F=29.35, P<0.0001) and treatment x time interaction =7.663, P<0.0001)  (Coc-Veh) CPP was significantly attenuated by an acute single injection of Ex4 (2.4 µg/kg) (Coc-Ex4) on the post-conditioning day compared to vehicle injection (The present study demonstrates that the physiological role of GLP-1 receptors extends beyond control of food intake and glucose homeostasis and includes reward regulation. Hence, we show for the first time that the ability of amphetamine and cocaine to induce a locomotor stimulation, accumbal dopamine release and to condition a place preference is reduced in mice treated peripherally with the GLP-1 analogue, Ex4.Albeit Ex4 was administered peripherally in the present study we hypothesize that the ability of Ex4 to attenuate the rewarding properties of psychostimulant drugs are mediated via GLP-1 receptors expressed within the mesolimbic dopamine system. Indeed, Ex4 readily passes the blood brain barrier . Moreoveper se on accumbal dopamine release, conditioned place preference and locomotor activity was used, and the selected dose should therefore not be anhedonic.Given that Ex4, at a dose that has no effect per se, reduces the amphetamine- as well as cocaine-induced reward we suggest that GLP-1 receptors have an important role in reward regulation. However, a similar dose of Ex4 caused a condition taste aversion , raisingper se, we hypothesize that GLP-1 receptors may play an important role in addiction processes. Supportively, Ex4, at a significantly higher dose, blocks the ability of amphetamine to cause a locomotor stimulation and cocaine to induce a CPP in rodents [The ability of addictive drugs to cause stimulation, dopamine release and to induce a CPP are intimately associated with the reinforcing properties of addictive drugs and these parameters are considered to constitute a part of the addiction process ,44–46. G rodents ,47. More rodents . The fin rodents  as well  rodents , imply t rodents . Indeed, rodents ,50 and a rodents ,52. More rodents ,54.per se, attenuates the psychostimulant-induced reward as measured by locomotor stimulation, accumbal dopamine release and CPP. Given that GLP-1 analogues, such as exenatide and liraglutide, are clinically available for treatment of type II diabetes, we propose that these should be elucidated as treatment of drug dependence.Our present findings show for the first time that Ex4, at a dose with no effect


P. vivax and P. falciparum malaria. This study compared the efficacy and safety of pyronaridine-artesunate with that of chloroquine for the treatment of uncomplicated P. vivax malaria.New antimalarials are needed for P. vivax mono-infection were randomized (1∶1) to receive pyronaridine-artesunate (target dose 7.2∶2.4 mg/kg to 13.8∶4.6 mg/kg) or chloroquine (standard dose) once daily for three days. Each treatment group included 228 randomized patients. Outcomes for the primary endpoint, Day-14 cure rate in the per-protocol population, were 99.5%,  with pyronaridine-artesunate and 100%  with chloroquine. Pyronaridine was non-inferior to chloroquine: treatment difference −0.5% , i.e., the lower limit of the 2-sided 95%CI for the treatment difference was greater than −10%. Pyronaridine-artesunate cure rates were non-inferior to chloroquine for Days 21, 28, 35 and 42. Parasite clearance time was shorter with pyronaridine-artesunate (median 23.0 h) versus chloroquine , as was fever clearance time . Kaplan-Meier estimates of post-baseline P. falciparum infection incidence until Day 42 were 2.5% with pyronaridine-artesunate, 6.1% with chloroquine . Post-baseline P. vivax or P. falciparum infection incidence until Day 42 was 6.8% and 12.4%, respectively . There were no deaths. Adverse events occurred in 92/228 (40.4%) patients with pyronaridine-artesunate and 72/228 (31.6%) with chloroquine. Mild and transient increases in hepatic enzymes were observed for pyronaridine-artesunate.This phase III randomized, double-blind, non-inferiority trial included five centers across Cambodia, Thailand, India, and Indonesia. In a double-dummy design, patients (aged >3–≤60 years) with microscopically confirmed P. vivax malaria was at least that of chloroquine. As pyronaridine-artesunate is also efficacious against P. falciparum malaria, this combination has potential utility as a global antimalarial drug.Pyronaridine-artesunate efficacy in acute uncomplicated NCT00440999Clinicaltrials.gov  Plasmodium vivax malaria, with up to 400 million clinical cases annually P. vivax malaria had been underappreciated; it can even be fatal P. vivax malaria live in the tropical areas across South and South East Asia, the remainder mostly live in the Western Pacific and Eastern Mediterranean P. vivax in areas where it had previously been eradicated, such as Korea and China, is of major concern An estimated 2.85 billion people are at risk of P. vivax treatment in most areas of the world for the past 50 years. However, P. vivax susceptibility to chloroquine has declined in some regions,  and new approaches are required urgently The blood schizontocide chloroquine and the tissue schizontocide primaquine have been the mainstay of Plasmodium vivax is highly sensitive to artemisinins P. vivax, artemisinin-based combination therapy (ACT) is recommended by The World Health Organization P. falciparum malaria P. vivax and P. falciparum are sympatric, ACT should have good efficacy (>95%) against both parasites, as differential diagnosis may not be feasible or reliable The blood stage of P. vivax and P. falciparum malaria. Pyronaridine is especially interesting as a component of ACT because in vitro studies using recent isolates indicate potent activity against chloroquine-resistant P. vivax and P. falciparumP. vivax similar to that of chloroquine P. falciparum malaria at Day 28 were >99%, and the combination was generally well tolerated Pyronaridine-artesunate is being developed as an ACT for use against P. vivax malaria. The primary objective of this clinical study was to compare the efficacy and safety of the fixed combination of pyronaridine-artesunate with that of standard chloroquine therapy in adults and children with acute, uncomplicated P. vivax malaria.This is the first pyronaridine-artesunate Phase III study reported for the treatment of The protocol for this trial and supporting CONSORT checklist are available as supporting information; see This study was conducted according to the principals of Good Clinical Practice and the Declaration of Helsinki and complied with all relevant regulatory requirements. The trial protocol was approved by institutional review boards in Cambodia , Thailand , India , and Indonesia . All patients or their guardians provided written informed consent and assent was required from children old enough to understand the study.This was a multi-center, randomized, double-blind, double-dummy, parallel-group comparative, non-inferiority trial conducted between March 2007 and March 2008 in local hospitals across five centers: Pailin, Cambodia; Mae Sot and Mae Ramat, Thailand; Mangalore, India; and Maumere (Island of Flores), Indonesia. There were no changes to the protocol after study commencement.P. vivax mono-infection confirmed by positive microscopy of P. vivax with a parasite density ≥250 µL−1 of blood  and fever or documented history of fever in the previous 24 h, and with a body weight between 20 and 90 kg  were eligible for enrollment. Patients were excluded from the study if they had: any other condition requiring hospitalization; anemia (hemoglobin <8 g/dL); hepatic or renal impairment; presence or history of clinically significant disorders; known hypersensitivity to study drugs or excipients; known active hepatitis A IgM, hepatitis B surface antigen, hepatitis C antibody or seropositive for HIV antibody; used an antimalarial within the previous two weeks (urine test required); used an antibacterial with anti-malarial activity within the previous two weeks; received an investigational drug within the past four weeks; previously participated in the study. Women who were pregnant or lactating were also excluded. Agreement to use an acceptable method of contraception during the study was required from all women of child-bearing age.Male or female subjects between three and 60 years of age with acute uncomplicated Study drugs were pyronaridine-artesunate tablets 180∶60 mg, chloroquine tablets 155 mg, and matching placebos . Study drugs were given orally with water once daily for three days  under direct observation of study staff. Vomiting of the first dose of study drug within 30 minutes of administration resulted in re-dosing. Vomiting of subsequent doses resulted in withdrawal from the study and treatment with rescue medication . For pyronaridine-artesunate, drug dose was based on body weight: 20–25 kg, 1 tablet; 26–44 kg, 2 tablets; 45–64 kg, 3 tablets; and 65–90 kg, 4 tablets, i.e. a pyronaridine-artesunate target dose of between 7.2∶2.4 mg/kg and 13.8∶4.6 mg/kg. The chloroquine dose for adults was 620 mg on Days 0 and 1 and 310 mg on Day 2. The chloroquine target dose for children was 10 mg/kg on Days 0 and 1 and 5 mg/kg on Day 2.All subjects remained hospitalized for Days 0–3, returning for follow-up assessments on Days 7, 14, 21, 28, 35 and 42. A medical history was taken at screening and physical examination performed at screening, Days 3, 28 and 42. Vital signs were monitored at screening, on Days 1, 2, 3 and 7 and at other visits if indicated. Clinical signs and symptoms of malaria were assessed at screening, Days 1, 2 and 3 and at other visits if indicated. Body temperature was taken every 8 h over at least 72 h following the first study drug administration or temperature normalization for at least two readings 7–25 h apart, then at each visit. Venous blood was drawn for hematology at screening, Days 3, 7, 28, and 42, and for clinical laboratory tests at screening, Days 3, 7, and 28, and Day 42 if clinically indicated. Urinalysis was performed on the same schedule. Adverse events were monitored throughout the study. 12-lead electrocardiograms were performed at screening, Day 2 and at Days 7, 14 and 42 if clinically indicated.Blood samples for testing glucose-6-phosphate dehydrogenase (G6PD) deficiency were collected on pre-printed filter paper. A semi-quantitative fluorescent spot test was used to distinguish between G6PD-deficient samples and those with normal or intermediate activity . Patients completing the study to Day 28 with normal G6PD activity were given a 14-day course of primaquine (15 mg/day for adults and 0.3 mg/kg/day for children) starting on Day 28 after all required assessments had been performed. G6PD-deficient patients completing the study to Day 28 were treated as per country policy.Plasmodium species, duplicate thin blood smears were taken at screening, pre-dose on Day 1 and at visits from Day 7 whenever parasitological blood samples were taken. Patients experiencing treatment failure were administered rescue antimalarial therapy as per local practice. All procedures scheduled for Day 28, including preparation of thin and thick films were performed before rescue medication was administered.Parasite density was determined by duplicate Giemsa-stained thick blood films examined independently by two microscopists; the mean of the two readings was recorded. In the case of discrepancy (i.e. >30%), a third microscopist reviewed the slides and the principal investigator made the final classification. Thick films were taken every 8 h until at least 72 h or until a negative smear was recorded at least 7–25 h apart, then at each visit, or if the patient returned unscheduled. Asexual parasite and gametocyte counts were recorded separately at screening; total parasite counts were recorded at all other assessments. Parasites were enumerated against 200 white blood cells (WBC) using 1,000× magnification. When the number of asexual parasites dropped below 10 per 200 WBC, counting was done against at least 500 WBC. A blood slide was considered negative if no asexual parasites were observed per 1,000 WBC. External quality control, was conducted by a central independent laboratory, blinded to treatment . All slides from patients with treatment failure were re-examined, plus the first 5 slides from each site and 5% of all subjects randomly. Results confirmed that procedures were being conducted as per the defined protocol. To determine P. vivax malaria.The primary objective of this clinical study was to compare the efficacy and safety of the fixed combination of pyronaridine-artesunate (180∶60 mg) with that of standard chloroquine therapy in adults and children with acute, uncomplicated P. vivax illness requiring hospitalization in the presence of parasitemia; or presence of parasitemia and axillary temperature ≥37.5°C any time between Days 3 and 14; or presence of parasitemia on any day between Days 7 and 14, irrespective of clinical condition. Subjects presenting with non-P. vivax malaria after clearance of P. vivax parasites were withdrawn from the study and given treatment for the new infection. If non-P. vivax malaria occurred on or after Day 14, the subject was considered a success for Day 14.Efficacy endpoints followed WHO 2002 guidelines for monitoring antimalarial drug efficacy, as were relevant at the time that the study was designed Secondary efficacy measures included: Day 28 cure rate; parasite clearance time, defined as the time from first treatment dose to aparasitemia for two consecutive negative readings taken between 7–25 h apart; fever clearance time, defined as time from first treatment dose to apyrexia for two consecutive normal temperature readings taken between 7–25 h apart; the proportion of patients aparasitemic on Days 1, 2, and 3; and the proportion of patients apyretic on Days 1, 2, and 3. The cure rates on Days 21, 35 and 42 were included as exploratory endpoints.Safety outcomes included: the frequency, nature and severity of adverse events; the frequency of serious adverse events; the frequency of adverse events considered by the investigator to be related to study medication; the reasons for study withdrawals; abnormal electrocardiograms; and abnormal hematology and clinical chemistry results. An adverse event was defined as any unfavorable and unintended sign, symptom, syndrome, or illness that developed or worsened during the period of observation in the clinical study. A serious adverse event was defined as one that resulted in: death or was life threatening; hospitalization or a prolongation of hospitalization; a congenital abnormality or birth defect; a persistent or significant disability or incapacity; or was judged by the investigator to be serious. Drug-related adverse events were determined by the investigator, based on the temporal relationship to drug therapy, any corroborating laboratory data, and improvement when drug was discontinued.Assuming a 90% cure rate on Day 14 in both treatment groups and a non-inferiority limit of −10%, a sample size of 410 evaluable subjects randomized in a 1∶1 ratio (205 subjects in each treatment group) would provide 90% power to demonstrate non-inferiority of pyronaridine-artesunate compared to chloroquine, using a 2-sided 95% confidence interval (CI). Assuming a dropout rate of 10%, 456 subjects would need to be randomized to the study (228 subjects in each treatment group). Assuming a Day-14 cure rate of 95%, this sample size would provide >99% power to demonstrate non-inferiority of pyronaridine-artesunate compared to chloroquine.A computer-generated randomization scheme was provided by the sponsor. Subjects were randomized 1∶1 within each study site in blocks of six to receive either pyronaridine-artesunate plus matching chloroquine placebo or oral chloroquine plus matching pyronaridine-artesunate placebo. Randomization numbers were assigned in ascending order to each subject according to the order recruited. The subject was allocated an individually numbered treatment pack, which contained sufficient tablets for 3 days' therapy plus an overage bottle containing tablets in case the subject vomited the first dose. Study drugs were administered on a double-blind, double-dummy basis. The investigator calculated the appropriate dose and study drug was administered by a different member of staff, designated by the investigator. All study investigators, laboratory technicians and patients were blind to treatment assignment. Active drugs and placebos were packaged similarly. Sealed opaque envelopes containing the study medication assignment for each subject were provided to the study site investigator for use in an emergency; no code breaks were required.The intent-to-treat (ITT) population was defined as all randomized subjects who received any amount of study medication. The per-protocol (PP) population included all randomized patients who completed a full course of study medication, had a known primary efficacy endpoint at Day 14 and did not violate the protocol in a way that would compromise efficacy evaluation, i.e. did not use prohibited concomitant medication; have a concomitant medical condition or infection that may have affected treatment outcome; or have any major protocol deviation.For this non-inferiority study, the primary efficacy endpoint of Day-14 cure rate was assessed in the PP population. Non-inferiority was demonstrated if the lower limit of the 2-sided 95% confidence interval (CI) for the difference in Day-14 cure rates was greater than –10%. The CI was calculated according to the Newcombe–Wilson method without continuity correction. Furthermore, exact (Pearson–Clopper) 95% CIs were calculated for the Day-14 cure rate in each of the two treatment groups. A similar analysis was repeated for the proportion of subjects with cure on Days 21, 28, 35 and 42.Parasite clearance time and fever clearance time were summarized using Kaplan-Meier estimates (log-rank test). Subjects without confirmed parasite clearance time or fever clearance time within 72 h after the first dose of study drug were censored at that time point.P. falciparum infection and for time to reappearance of any parasite  were also conducted using Kaplan-Meier estimates. All statistical evaluations were performed using SAS®, Version 9.1.3 in a UNIX environment.Post-hoc analyses of time to P. falciparum infection compared with 5/228 (2.2%) in the pyronaridine-artesunate group. More patients were excluded from the PP population in the chloroquine group, 19/228 (8.3%), than the pyronaridine-artesunate group, 10/228 (4.4%), mainly because of missing efficacy data and insufficient drug therapy.Patient disposition is shown in Patient baseline demographic and clinical characteristics are shown in Outcomes for the primary endpoint, cure rate at Day 14 in the PP population, were 99.5%,  in the pyronaridine-artesunate group and 100%  in the chloroquine group. Pyronaridine was non-inferior to chloroquine for the primary endpoint: treatment difference −0.5% . All 27 children ≤12 years were cured at Day 14. Day-14 cure rates were 100% in all study centers, except for the one failure in the pyronaridine-artesunate group, which occurred in a patient from Maumere, Indonesia . Results for Day-14 cure rate in the ITT population were supportive of the primary analysis; 95.2%;  with pyronaridine-artesunate and 93.0%  with chloroquine, treatment difference 2.2% . At all efficacy assessments from Day 14 until Day 42, pyronaridine-artesunate was non-inferior to chloroquine, with cure rates >95% .Using Kaplan–Meier analysis of the ITT population, time to parasite clearance was shorter in the pyronaridine-artesunate group compared with the chloroquine group  patients in the pyronaridine-artesunate group and 179/228 (78.5%) patients in the chloroquine group. Therefore, evaluation of the anti-pyretic activity of pyronaridine-artesunate and chloroquine cannot be fully assessed. However Kaplan-Meier analysis of the ITT population showed a shorter time to fever clearance with pyronaridine-artesunate than with chloroquine  patients experienced a treatment-emergent adverse event of any cause compared with 72/228 (31.6%) in the chloroquine group . IncreasThere were no deaths during the study. Two patients, both in the pyronaridine-artesunate group, had serious adverse events ; neither was considered drug related by the investigator. Two patients, both in the chloroquine group, had adverse events leading to drug discontinuation and study withdrawal ; all considered possibly related to drug treatment by the investigator.Baseline hemoglobin levels were similar in the two treatment groups . A transBiochemistry laboratory observations were generally similar in both treatment groups, with the exception of alanine transaminase (ALT) and aspartate transaminase (AST) . From DaClinically significant electrocardiograms were recorded for 1/226 (0.4%) patients in the pyronaridine-artesunate group and 6/222 (2.7%) in the chloroquine group. All cases were recorded as QTc prolongation. All except one event (in the chloroquine group) was considered unrelated to study treatment by the investigator.P. vivax malaria with a Day-14 cure rate of 99.5% that was non-inferior to that of chloroquine (100%). Non-inferiority of pyronaridine-artesunate to chloroquine was maintained throughout the study. However, primaquine treatment was initiated in most cases after Day 28, making comparisons after this time point difficult. There are no comparative data of pyronaridine-artesunate in P. vivax malaria. Results for chloroquine were comparable to other studies in Thailand and India (100% efficacy) P. vivaxPyronaridine-artesunate was highly efficacious in the treatment of uncomplicated P. vivax malaria have also demonstrated rapid clearance of parasites and fever The clinical efficacy of the artemisinin derivatives is characterized by an almost immediate onset of activity and rapid reduction of parasitemia P. vivax or P. falciparum post-baseline infection was lower with pyronaridine-artesunate versus chloroquine, mainly because of the difference in P. falciparum post-baseline infections. Post-baseline infection with P. falciparum was less frequent and occurred later in the study with pyronaridine-artesunate versus chloroquine. Given the potential severity of P. falciparum malaria, this difference is clinically important. P. falciparum chloroquine resistance is widespread in vitro activity against chloroquine-resistant P. falciparumP. falciparumP. falciparum infections in the chloroquine group. However, we cannot confirm this as susceptibility or molecular testing of isolates was not performed.The incidence of P. falciparum malaria Adverse events of any cause with pyronaridine-artesunate in this study were consistent with reports for pyronaridine and artemisinins as monotherapy in falciparum malaria P. vivax and P. falciparum infection and P. falciparum reinfection are common in areas endemic for P. vivax. Practical difficulties in reliably distinguishing between the two pathogens means that, in practice, many malaria cases will be diagnosed clinically and treated empirically P. vivax/P. falciparum infection were excluded. However, our design does reflect the clinical situation in the study region where known P. falciparum malaria would not be treated with chloroquine because of the high risk of failure from widespread resistance to this agent P. vivax and mixed P. vivax/P. falciparum infection and would compare pyronaridine-artesunate to another ACT.Mixed P. vivax chloroquine resistance. In vitro data indicate high activity for pyronaridine against chloroquine-resistant P. vivaxIn this Phase III trial, the study population was restricted in order to allow comparison of the two treatments and probably does not reflect the baseline clinical and demographic characteristics of the general population of malaria patients in the region. Further studies are required to assess pyronaridine-artesunate in the wider clinical setting. Further data are also required to assess pyronaridine-artesunate efficacy in areas of high P. vivax malaria, particularly because this group is the most vulnerable to severe outcomes Our study included only 27 children, all over 7 years of age (probably because of the 20 kg minimum weight requirement). Pyronaridine-artesunate efficacy and safety data are required in very young children with P. vivax. Post-baseline P. falciparum infection was reduced and delayed with pyronaridine-artesunate versus chloroquine. Based on the results of this study in P. vivax and those conducted against P. falciparumIn conclusion, pyronaridine-artesunate had non-inferior efficacy and faster parasite and fever clearance compared with chloroquine against Checklist S1CONSORT checklist.(0.23 MB DOC)Click here for additional data file.Protocol S1Trial Protocol.(0.62 MB PDF)Click here for additional data file.


Many genetic association studies used single nucleotide polymorphisms (SNPs) data to identify genetic variants for complex diseases. Although SNP-based associations are most common in genome-wide association studies (GWAS), gene-based association analysis has received increasing attention in understanding genetic etiologies for complex diseases. While both methods have been used to analyze the same data, few genome-wide association studies compare the results or observe the connection between them. We performed a comprehensive analysis of the data from the Study of Addiction: Genetics and Environment (SAGE) and compared the results from the SNP-based and gene-based analyses. Our results suggest that the gene-based method complements the individual SNP-based analysis, and conceptually they are closely related. In terms of gene findings, our results validate many genes that were either reported from the analysis of the same dataset or based on animal studies for substance dependence. P value. Then the top SNPs were mapped into functional regions such as a gene or pathway to facilitate further investigation of the corresponding gene and disease. Based on SNP-based association analysis, many genetic variants underlying complex diseases or traits were detected [P value <5 × 10−8 is usually used as the threshold to declare a genome-wide significance SNP, which also limits the discoveries of the genes that are important to the disease. Also importantly, susceptible SNPs generally explain a small fraction of the risk—a phenomenon commonly referred to as the “missing heritability” [Genome-wide association studies (GWAS) have become a powerful tool in the identification of susceptible loci for numerous diseases . A typicdetected , 3. Due detected . A predeability” . To alleability” –9, whichability” , which eGene-based methods have been successfully applied to GWAS of complex diseases, including Crohn's disease , type 1 Recent studies show that there are many candidate genes associated with substance dependence. For example, GABRA2, CHRM2, ADH4, PKNOX2, GABRG3, TAS2R16, SNCA, OPRK1, and PDYN are well studied for alcohol addiction and have been replicated in many samples –28. HoweBased on the analysis of the SAGE data, we report a number of susceptible loci at the SNP and/or gene levels, which validate many susceptibility loci that have been reported to be associated with substance dependence , 40–44. r2 > 0.9) with the initially assigned SNPs within the gene [The dataset included 4,121 subjects in SAGE with six categories of substance dependence data: alcohol, cocaine, marijuana, nicotine, opiates, and other dependencies on drugs. The data were downloaded from dbGaP (study accession phs000092.v1.p1) . SAGE 1 is a larthe gene . TogetheE − 8 and 2.5E − 6 as the genome-wide significant thresholds for SNP-based and gene-based methods, respectively [P < 1.0E − 5 and genes P < 5.0E − 4 were considered further. These P values are referred to as relaxed significance thresholds below. The selected SNPs were then mapped into the corresponding genes by the mapping rule proposed above.Following the conventional standards, we used 5.0ectively . To incrP value of each SNP was evaluated by the logistic regression, and then the correlation coefficients (r2) of all SNP pairs were calculated. The computation was performed in PLINK software (version 1.07) [m SNPs are assigned to a gene; an association test such as through the traditional logistic regression or linear regression is used to examine the association between the phenotype and each single SNP. This step yields mP values for m SNPs. GATES combines the available mP values within a gene by using a modified Simes test to give a gene-based P value. The summary P value is defined aspj) is the effective number of independent P values among the top j SNPs. The effective number of independent P values was derived by accounting for the LD structure among the specified SNPs; we refer to [We took several steps in testing the associations between genetic variants (SNP or gene) and substance dependenice. First, the on 1.07) . In the on 1.07)  based onP values were less than 1.0E − 5 and then mapped them into the corresponding genes. This allows us to compare the susceptible genes identified by both methods discussed above.In order to compare the performance of the SNP-based and gene-based methods, in the SNP-based method, we selected those SNPs whose P  values for the gene-based method, the minimal P  value of SNPs within a gene, and their literature references and reported substance dependence. Next, we performed a literature search on the genetic regions which contain the identified genes and filtered the susceptible genetic regions which have been reported to associate with substance dependence for further investigation. In P values, −log10(P). Each point for the SNP-based analysis in P value within the gene.In P = 3.25E − 5), rs9610271 (P = 4.19E − 5), rs9610417 (P = 5.38E − 5), and rs2876981 (P = 7.51E − 5). The P values for these SNPs are slightly greater than the relaxed SNP-based threshold (P < 1.0E − 5), and hence the SNP-based method failed to detect them. Overall, five genes, NCK2 (opiates dependence in black men), SH3BP5 (cocaine dependence in white men), LRP5 (opiates dependence in white men), KIAA0040 , and PKNOX2 , were identified by both the SNP-based and gene-based methods as meeting either of the relaxed significance levels for a specific dependence and within a gender-racial group. Four genes, MAPK1 (marijuana dependence in black women), MANBA , HAAO (cocaine dependence in white women), and IFNG (opiates dependence in white women), met the threshold by the gene-based method only. We found that the significant signal of gene MAPK1 was mainly driven by SNPs: rs7290469 . For CSMD1, 1,934 SNPs were mapped into it. Its signal was mainly determined by only five SNPs: rs2624087 , rs4875371 , rs2623607 , rs10503267 , and rs4875372 . Because there were only 5.3% of the SNPs (103 SNPs) with P  value less than 0.05, the overall association from the gene became less significant.Furthermore, four other genes, FAM38B (cocaine dependence in black women), PTPRM (marijuana dependence in black women), CSMD1 (nicotine dependence in black women), and RELN (cocaine dependence in white men), contain at least one SNP that met the SNP-based relaxed threshold of significance. The gene-based Since none of the SNPs attained the genome-wide significance for any dependence by the SNP-based method, in this section we will only focus on the results from the gene-based method.P value  <1.0E − 5. This method identified one genome-wide significant gene, DSG3  for nicotine dependence in white men. The P value of gene NCK2: 2.70E − 6 is very close to the genome-wide significant threshold, which provided very strong evidence for the association of opiates in black men. As shown in P value = 1.09E − 7) for NCK2 gene and rs6701037  and rs1057302  for DSG3 gene. However, none of these SNPs reached genome-wide significance.In this paper, we thoroughly analyzed the SAGE data from the SNP-based and gene-based methods, and compared the results obtained from these two methods. Specifically, for each sex-racial group, we performed association analysis for the six categories of substance dependence separately. The gene-based method appears to be more powerful in detecting susceptibility loci. Most of the genes identified in our study are supported by various reports in the literature related to the genetics of substance dependence , 48. BasP = 2.70E − 6) associated with nicotine dependence in the white men, according to the gene-based method. Another gene, NCK2, is nearly genome-wide significant (P = 2.7E − 6) in its association with substance dependence. Overall, we did not detect any genome-wide significant SNP when using the SNPs-based method. However, one gene, DSG3,is genome-wide significantly (P value of the SNPs within a gene, whereas the typical gene-based method uses a weighted approach. The advantages and limitations of these two approaches are similar to those between the extreme function and a weighted average.The SNP-based method and gene-based method are closely related. In fact, the SNP-based method can be viewed as a gene-based method using the extreme function, namely, the minimal We should point out that both the SNP-based and gene-based methods have their own advantages and disadvantages. The SNP-based method has its unique strength in identifying genes with only a small number of significant SNPs. However, since the SNP-based method focuses on a single SNP at a time, it is less powerful to detect a gene whose SNPs have weak marginal effects, but a strong joint effect. In our analysis, 207 genes passed the relaxed gene-based threshold, whereas only 64 genes passed the relaxed SNP-based threshold.  Both the SNP-based and gene-based methods can be conducted conveniently in commonly available software, such as PLINK  for the


Background. Advanced basal cell carcinoma (BCC) is often treated by surgery or X-ray therapy. The consequences of X-ray therapy on the patients' health-related quality-of-life (HRQOL) have so far not been described. Objectives. To quantify quality of life in BCC patients before and after X-ray therapy compared with matched healthy controls. Materials. Twenty-five patients (mean age 69) with BCC completed the Dermatology Life Quality Index (DLQI) before and two weeks and three months after X-ray therapy and their results were compared with the DLQI scores for 25 matched controls. Results. Compared to the healthy controls the patients' DLQI score was significantly higher before and 2 weeks after X-ray therapy . The patients' DLQI score decreased significantly from baseline to three months after X-ray therapy (P = 0.024), when it became similar to that of the healthy controls (P = 0.819). Three months after X-ray therapy eight patients had no skin reactions, 11 had slight atrophy, pigmentation change, and/or some hair loss, four had patch atrophy, moderate telangiectasia, and/or total hair loss. Conclusions. BCC has a negative effect on patients' quality of life. The study shows that HRQOL normalises shortly after X-ray therapy, despite minor skin manifestations. Nonmelanoma skin cancers include basal cell carcinoma (BCC), squamous cell carcinoma, and more uncommon entities such as Merkel cell carcinoma , 2. BCC Unlike many other medical conditions, skin disorders are visible and may result in recognition of illness and possible stigmatization. Approximately 80% of BCCs occur in the head and neck and may cause disfiguring skin changes with impact on the patient's body image and health-related quality-of-life (HRQOL) .Two studies have used the Dermatology Life Quality Index (DLQI) to assess HRQOL in patients with BCC who have been treated with cryotherapy, curretage, excision, and Mohs for BCC surgery , 6, but The aim of the present study was to quantify the HRQOL in patients treated by X-ray for BCC.The study population consisted of 25 patients with BCC treated by X-ray  in 10 fractions over two weeks) and 25 healthy controls without skin diseases matched by gender and age . The conTo assess the specific impact of X-ray therapy, the validated short, self-administered questionnaire DLQI was used –7. The DThe study protocol was approved by the Danish Data Protection Agency (CVR-nr. 11-88-37-29) and the Scientific Ethical Committee for Copenhagen County (KF 01 271885) and was conducted in accordance with the Declaration of Helsinki V. All participants gave written informed consent. U-test. The different effects of the X-ray therapy on DLQI at the different times were analysed using the Wilcoxon two-sample test. A two-sided P value <0.05 was considered statistically significant.Data were analysed in SPSS Version 19.0 for Windows . The comparison of the patients with BCC and the healthy controls was performed using the Mann-Whitney n = 25) and confirmed by a pathologist.Twenty-five patients with BCC  and 25 healthy controls matched by gender and age participated in the study. Twenty-one (84%) of the BCCs were located on the head or neck, 3 (12%) on the trunk, and 1 (4%) on a leg. The lesion diameters at baseline were 7–60 mm (mean 25.9 mm) and had been present for a mean of 27 months (range 6–84 months) before X-ray treatment. The BCC diagnoses had in all cases been ascertained by a dermatologist (P = 0.024) and from two weeks to three months after X-ray therapy (P = 0.002).At baseline three (12%) patients had an ulcerative BCC. Eight (32%) patients had ulcerations immediately after the X-ray and 21 (84%) patients subsequently developed radiotherapy induced ulcerations, which had healed three months later. Three months after X-ray therapy nine patients had no skin reactions  , 12 had P = 0.004). Generally the scores in each of the subcategories and the overall scores of DLQI were very low  (P = 1.000).Scores in the subcategory “symptoms and feelings” significantly decreased from two weeks to three months after X-ray therapy (very low . The pat= 0.001) . Three mThis study demonstrated a significant effect on patients' HRQOL from baseline to three months after therapy .HRQOL is considered an important outcome in cancer research, and is associated with sequela such as depression, anxiety, and vulnerability . AlthougIn the present study the overall DLQI score was between 0.4–1.5 and this represents a very small effect on patient's HRQOL according to Finlay and Khan Tables  and 3 77. The meDLQI has been used in two other studies in relation to surgery for BCC  5, 6]. , 6. 5, 6Within the first month X-ray therapy acute reactions are common, which may be reflected in DLQI's subcategory “symptoms and feelings”. Concern about the acute side effects of X-ray therapy seems not to be a problem after X-ray therapy , as the The decline of the DLQI could be a result of confounders, but except for instructions in sun protection and care of ulcers, there was no difference between the healthy and the intervention group. Patients with BBC located at visible areas such as face or neck may suffer more than patients with BCC on at trunk and legs. This is confirmed by a separate analyse of face elements showing a significant difference in DLQI from before to 2 weeks after treatment, different from the total material.A number of authors argue that DLQI may be too non-specific for this use –14. The The present study shows that for 14 out of 25 patients more than a year passed before the patients sought medical advice, and the same tendency is seen in other studies . This coIn conclusion, the diagnosis, BCC, has a negative effect on patients' HRQOL, and cure by X-ray therapy brought HRQOL levels back to those of healthy controls, despite minor skin manifestations.


Orexin A  is a newly discovered potential analgesic substance. However, whether OXA is involved in acupuncture analgesia remains unknown. The present study was designed to investigate the involvement of spinal OXA in electroacupuncture (EA) analgesia.A modified rat model of post-laparotomy pain was adopted and evaluated. Von Frey filaments were used to measure mechanical allodynia of the hind paw and abdomen. EA at 2/15 Hz or 2/100 Hz was performed once on the bilateral ST36 and SP6 for 30 min perioperatively. SB-334867, a selective orexin 1 receptor (OX1R) antagonist with a higher affinity for OXA than OXB, was intrathecally injected to observe its effect on EA analgesia.OXA at 0.3 nmol and EA at 2/15 Hz produced respective analgesic effects on the model (P<0.05). Pre-surgical intrathecal administered of SB-334867 30 nmol antagonized OXA analgesia and attenuated the analgesic effect of EA (P<0.05). However, SB-334867 did not block fentanyl-induced analgesia (P>0.05). In addition, naloxone, a selective opioid receptor antagonist, failed to antagonize OXA-induced analgesia (P>0.05).The results of the present study indicate the involvement of OXA in EA analgesia via OX1R in an opioid-independent way. Acupuncture has been used in China since ancient times to relieve many different types of pain. This approach has few side effects and is considered to be a new, effective alternative medicine in Western countries. However, the mechanism underlying acupuncture analgesia remains unclear. Although the opioid system is definitely involved in acupuncture analgesia, naloxone, an opioid receptor antagonist, can only partially block the analgesic effect of acupuncture -4. Also,Discovered in 1998, orexin  encodes a hypothalamic neuropeptide precursor protein that gives rise to two mature neuropeptides, orexin A (OXA) and orexin B (OXB), by proteolytic processing. Hcrt mRNA is mainly expressed in the lateral and perifornical areas of the posterior hypothalamus ,14. OXA To date, there are few reports on the function of OXA in acupuncture analgesia. Whether CNS OXA levels change after surgery or if OXA is involved in electroacupuncture (EA) analgesia remains unknown. The present study was designed to monitor OXA changes post-surgery and its role in EA analgesia. Using a modified post-laparotomy pain model, which is more closely related to clinical use, the present study was intended to (1) examine the change in OXA levels both in the rat model of post-laparotomy pain and after EA treatment of the model; (2) evaluate the modified rat model of post-laparotomy pain and observe the analgesic effect of intrathecal injected OXA and EA application on the model; (3) test the inhibitory effect of the selective OX1R antagonist, SB-334867, on OXA and EA analgesia; and (4) further verify whether OXA analgesia was independent of the opioid system in the rat model of post-laparotomy pain.A rat model of post-operative pain  was adopad libitum. All protocols for this study were approved by the Animal Care and Use Committee from Shanghai Medical College, Fudan University, and the experiments were conducted in accordance with IASP guidelines for pain research in animals [Adult male Sprague–Dawley rats (200–220 g) were purchased from the BK Company  and allowed to acclimate for one week prior to the beginning of experiments. To minimize possible disturbances, the animals were housed in a quiet room close to the operating room with a 12:12-h light/dark cycle. Room temperature was maintained at 20 ± 2°C. Food pellets and water were supplied  animals .Animals tend to explore new environments. This may affect their sensitivity to external stimuli . To miniA rat model of laparotomy pain was established according to a previous report  and was (Zusanli) points to a depth of 5 mm and SP6 (Sanyinjiao) points to a depth of 3 mm. The stimulator was turned on, and the frequency was adjusted to 2/15 Hz or 2/100 Hz, while the current was adjusted to 1–3 mA. At this current, slight shaking of the legs was induced. The time course of EA application was 30 min, and the operation was conducted at 10 min for approximately 10 min. For rats in the sham EA group, needles were inserted into the bilateral ST36 and SP6 points with no current stimulation.For animals in the groups that received EA stimulation, EA was applied via two stainless steel needles (0.3 mm in diameter and 25 mm long) connected with the output terminals of an EA apparatus, the HANS Acupoint Nerve Stimulator . The needles were vertically inserted into bilateral ST36 Rats in the four groups  were sacrificed 1 h after surgery or EA. Samples were collected from the hypothalamus, PAG and spinal cord (T13-L5). Brain regions were dissected according to conventional definitions . PeptideDried samples stored at −80°C were used for OXA measurements, which were quantified using the standard ELISA kit (EK-003-30) obtained from Phoenix Pharmaceuticals Inc.  according to the manufacturer instructions. ELISA was chosen to minimize the usage of radioactive materials and, both ELISA and RIA methods used to quantify orexin have been evaluated and used ,37. OptiFentanyl citrate, an opioid μ-receptor agonist and a clinically used analgesic, and the control  were intramuscularly (i.m.) injected. SB-334867 , naloxone  or OXA  were intrathecally injected. Identical volumes of drug vehicle were administered for comparison with drug-treated groups in each experiment.For intrathecal injections, a modified method of the direct transcutaneous intrathecal injection was used . After bThe von Frey filaments  were used to identify the severity and duration of mechanical allodynia state in rats according to previously published literature ,40. We iACT and PWT were tested consecutively. Filaments were applied from underneath the chamber through openings in the mesh floor to an area 5 mm adjacent to the abdominal wound line (ACT) or to an area in the plantar surface of the left foot (PWT). The filaments produced an upward force to induce abdominal constriction or paw withdrawal. A preliminary experiment was conducted to select the proper sequential filaments, as shown in Table Each of the sequential von Frey filaments was applied once for 3 s starting with the minimum and continuing until an abdominal constriction or foot withdrawal response occurred. After a 3-min test-free period, each filament was again applied once, beginning with the minimum, until a response was elicited. This was repeated a third time 3 min later. The lowest force from the three tests producing a response was considered the ACT or PWT. The cutoff value, 26.0g, was recorded even if there was no response to this force. The animals were tested at baseline, 0.5, 1, 2, 4, 6 and 24 h after surgery. Time 0 was defined as the end of the surgery or EA application.The treatment regimes were:(a) No drug (or EA) treatment:normal group, model group (rats undergone surgery) and sham model group (anesthesia only rats) (n=8 per group).Post-operative treatment with fentanyl:To fully evaluate the pain state of the laparotomy model, rats received a fentanyl injection  or drug-vehicle  immediately after surgery at time 0.(b) Post-operative treatment with OXA:To verify whether OXA has an analgesic effect on the model, rats received intrathecally administered OXA  or drug-vehicle  after surgery at time 0.Perioperative treatment with EA:To explore the EA analgesic effect on the rat model, EA was applied perioperatively for 30 min, i.e., EA stimulation (2/15 Hz or 2/100 Hz) begun 10 min before surgery and ended 10 min after surgery. During the middle 10 min of EA the surgery was completed. Thus, when EA was applied in the experiment, time 0 was defined as the end of EA.(c) Pre-operative treatment with SB-334867on OXA analgesia:To explore whether OXA produces its analgesic effect via OX1R, the selective OX1R antagonist SB-334867  or drug-vehicle  was intrathecally administered prior to surgery OXA with the minimum effective dose selected from (c) was intrathecally administered post-surgery.Pre- or post-EA treatment with SB-334867:To examine whether OXA participates in EA analgesia via OX1R, the selective OX1R antagonist SB-334867  or drug vehicle (DMSO 10 μL) was intrathecally administered prior to or post-EA-treatment.(d) Pre-EA treatment with naloxoneTo explore whether OXA analgesia was independent of the opioid system, the opioid receptor antagonist naloxone 28 nmol or drug vehicle  was intrathecally injected before surgery OXA, was intrathecally injected after surgery.Statistical analyses were performed using SPSS 13.0 software. Threshold response data were represented as the mean ± standard error of means (SEM). Between groups, ACT and PWT data at each time point were analyzed by a one-way analysis of variance (ANOVA) followed by a post hoc least significance difference test (LSD). A probability (p) value of < 0.05 was considered to be statistically significant.The ELISA tests showed that there was a significant decrease of OXA peptide levels in the hypothalamus, PAG and spinal cord in rats after surgery (P<0.05) when compared with the normal group. EA 2/15 Hz reversed the decrease in OXA levels in the above regions in model rats (P<0.05), whereas the EA2/100 Hz group showed no change .No significant difference in mechanical thresholds was detected between the normal and sham model group (P>0.05), as shown in Figure The μ-opioid receptor agonist fentanyl was intramuscularly (i.m.) injected immediately after surgery at 5, 10 and 20 μg/kg. Figure OXA at a concentration of 0.1, 0.3 and 1.0 nmol or saline was intrathecally injected into animals immediately after surgery. Figure Perioperative EA stimulation was applied bilaterally to the ST36 and SP6 acupoints at 2/15 Hz or 2/100 Hz for 30 min. Surgery was performed during the middle 10 min of EA stimulation. Figure The OX1R antagonist SB-334867 (30 nmol) was intrathecally injected prior to surgery. Figure SB-334867 was intrathecally injected into the rat model in addition to EA application. SB-334867 (30 nmol) had no obvious effect on the ACT at 0.5 h when compared with the DMSO group (P>0.05 by one way ANOVA with LSD test). Pre-surgical treatment with SB-334867 (SB-pr) significantly decreased EA analgesia (P<0.05 by one way ANOVA with LSD test)  post-surgery. At 0.5 h post-OXA injection, the OXA 0.3 nmol group and the OXA + Naloxone group both showed a significant difference in the ACT when compared with the saline group (P<0.05), whereas there was no difference between the OXA group and the OXA + Naloxone group (P>0.05) Figure A.In another experiment, we intrathecally injected SB-334867 pre-surgery followed by an intramuscular injection of fentanyl post-surgery. Results showed there was no significant difference between the SB-334867 group and model group, or between the SB-334867 + fentanyl group and fentanyl group , at a dose that blocked dynorphin-induced analgesia, was unable to block OXA-induced analgesia. This result is in accordance with previous studies ,29. AlsoIn addition, OXA levels in the hypothalamus, PAG and spinal cord also decreased significantly after laparotomy and were reversed by EA. The above areas are included in the descending inhibitory system. This result has shown that EA can regulate the descending inhibitory system to achieve an analgesic effect. We can only infer that the possible reason for an OXA decrease in the model group is that the laparotomy is a traumatic injury to rats and induces a stress response, which causes exhaustion of OXA in the CNS. OXA is produced through hydrolysis of its precursor PPO by the proteolytic enzyme during the process of axonal transport . The mecAlthough orexin A (OXA) is a newly discovered potential analgesic substance, whether OXA is involved in acupuncture analgesia remains unknown. In this study we have shown that EA reversed the OXA decrease in rats post laparotomy. Intrathecal administered of OX1R antagonist SB-334867 antagonized OXA analgesia and attenuated the analgesic effect of EA, but did not block fentanyl-induced analgesia. In addition, naloxone failed to antagonize OXA-induced analgesia. The results of the present study indicate the involvement of OXA in EA analgesia via OX1R in an opioid-independent way.The authors declare that there are no conflicts of interest to declare.XMF WLM and FX carried out the study design, experimental work, data collection and interpretation, literature review, and manuscript preparation. YQW and QLMY provided assistance with applying for the supporting grant. JWJ provided an excellent research environment and participated in discussion and coordination. GCW supervised the work, evaluated the data, and corrected the manuscript for publication. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/225/prepub